A cold-chain free loop-mediated isothermal amplification test for detection of human malaria in point-of-care settings.

Malaria diagnosis in low-resource settings demands robust and cold-chain free molecular tests. To overcome infrastructure limitations of conventional loop mediated isothermal amplification (LAMP), we developed a refrigeration-independent assay for Plasmodium knowlesi detection. By using sugar-based enzyme preservation approach, sucrose shows most effectively to maintain Bst DNA polymerase activity during room-temperature storage, outperforming equivalent concentrations of glucose and galactose. The sucrose-preserved LAMP reagent demonstrated consistent performance for 12 days. This optimized assay achieved analytical sensitivity (10 copies/μL) and maintained 100 % specificity against Plasmodium species and human DNA. The chemical preserving method provided a cost-effective alternative to lyophilization, eliminating dependence on specialized equipment while achieving comparable short-term stability. The 12-day room-temperature stability suits typical field deployments, making the assay ideal for malaria surveillance and use in remote clinics.